Molecular Cloning of Platelet Factor XI, an Alternative Splicing Product of the Plasma Factor XI Gene

Abstract

Platelet factor XI is associated with the platelet plasma membrane and has an apparent M r (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of ϳ1.9 kilobases was detected in megakaryocytic cells, and one of ϳ2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala 91 -Arg 144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.Plasma coagulation factor XI is a glycoprotein present in human plasma at a concentration of ϳ30 nM as a zymogen that, when converted by limited proteolysis to an active serine protease, participates in the contact phase of blood coagulation. This zymogen is an unique plasma coagulation enzyme because it exists as a homodimer (M r ϳ143,000) consisting of two identical polypeptide chains linked by disulfide bonds (1, 2).The sequence of the human factor XI gene has been elucidated by sequencing of the cDNA inserts coding for factor XI from two different phage genomic libraries (3,4 Factor XI coagulant activity and antigen are present in wellwashed platelet suspensions, and the activity accounts for about 0.5% of the total factor XI activity in blood (5, 6). About half of factor XI-deficient patients have defective hemostasis, whereas the remainder do not experience abnormal bleeding even in the absence of plasma factor XI (7). Subcellular fractionation studies have shown that factor XI activity is enriched in the platelet membrane fraction (8). The washed platelets and isolated platelet membranes obtained from a factor XIdeficient donor without a history of excessive bleeding had normal quantities of factor XI-like activity and normal behavior in the contact phase of coagulation (9). Thus, the functional significance of factor XI associated with platelets is not clear, but it may play a role in maintaining normal hemostasis, possibly complementing plasma factor XI deficiency (9).Previous studies employing immunofluorescence, immunoelectrophoresis, and immunoprecipitation utilizing monospecific polyclonal anti-factor XI antibodies have demonstrated the presence in and partial purification from human platelets of a molecule ...

Compound heterozygosity for two novel mutations in a severe factor XI deficiency

“…In this patient, platelet expression of the truncated FXI molecules was not studied, but both mutations generate an FXI molecule in which the catalytic domain is completely disrupted and thus may not be functionally present in platelets. At present, the patient's phenotype is not fully explained by FXI gene analysis, but it is possible that unidentified FXI-like molecules on platelets could be induced and thus compensate for the absence of plasma FXI as described previously [21][22][23]. …”

Characterization of the H-kininogen-binding Site on Factor XI

“…Recently, a splice variant of FXI mRNA lacking the sequence encoded by exon 5 of the FXI gene (coding for amino acids Ala 91 -Arg 144 in F2) has been described (28). Flow cytometry studies indicate that this variant is expressed on platelet membranes independent of plasma FXI expression and thus may compensate partially for plasma FXI deficiency (29).…”

“…The results indicate that the binding interaction between FXI and HK is similar to the interaction between PPK and HK, involving multiple apple domains, with the second apple domain being most important. We tested these findings with additional experiments on a recently identified FXI splice variant lacking the N-terminal half of F2 (28) that is present in platelets (29). This protein has greatly reduced HK binding compared with full-length FXI with potential (patho)physiological consequences.…”

Molecular characterization of in-frame and out-of-frame alternative splicings in coagulation factor XI pre-mRNA

“…[21][22][23] Concerning the F11 gene, the existence of AS isoforms has been known for many years. 6,10,26 Unfortunately, information on the splicing pattern of this gene is still unclear, if not misleading. Indeed, most nucleotide and protein databases still report the existence of a "FXI isoform 2," lacking amino acids from 91 to 144, 10,37 notwithstanding that this isoform was not confirmed by more recent literature 5,25 and the relevant reference sequence (NM_019559.2) was removed from GenBank (National Center for Biotechnology Information) because of insufficient supporting data.…”

“…[7][8][9] The site of synthesis and nature of this platelet variant still remain a matter of debate. 6,10 The existence of a platelet transcript, originating from the skipping of exon 5, was thought for a long time to account for the "platelet-specific" FXI isoform, 10 even though the not-in-frame nature of this exon skipping, and the subsequent introduction of a premature termination codon (PTC), was not easily reconcilable with translation after exon 5. Indeed, subsequent studies did not confirm the presence of an alternatively spliced transcript lacking exon 5 in human platelets, in which it was only possible to detect full-length correctly spliced FXI mRNA.…”